Antibody-mediated glomerular injury is a major cause of glomerulonephritis leading to loss of renal function. Type IV collagen of the GBM is the target of pathogenic antibodies in Goodpasture's (GP) disease, Alport post-transplant nephritis, and experimental anti-GBM disease. Antibodies bind to the NC1 domains of the alpha3alpha4alpha5(IV) network, which are present in the native GBM as a hexamer complex. The recent mapping of the cryptic GP epitopes within the alpha3(IV) NC1 domain and the structural characterization of the alpha3alpha4alpha5(IV) NC1 hexamer set the stage for addressing new questions about the molecular pathogenesis of anti-GBM disease. How GP autoAbs bind to their epitopes, sequestered in the NC1 hexamers in the native GBM, and how the cryptic epitopes could be exposed to initiate an auto-immune response will be established. The location of the epitopes of Alport alloAbs will be mapped. The specificity of anti-GBM antibodies in animal models of anti-GBM nephritis, and whether they resemble GP autoAbs or Alport alloAbs, will be determined. The central hypothesis is that the etiologic agents and the targets of pathogenic anti-GBM Abs are: (a) cryptic epitopes, sequestered within the native NC1 hexamers of GBM, in GP disease; but (b) exposed epitopes, accessible in the NC1 hexamers, in Alport post-transplant nephritis. The rationale is that immune tolerance toward native GBM components restricts the repertoire of anti-GBM Abs to cryptic epitopes in GP disease, but not in Alport syndrome, in which the GBM alpha3alpha4alpha5(IV) network is absent. Four specific hypotheses will be tested: (1) Pathogenic GP autoAbs, unlike Alport alloAbs, bind only to a sub-population of NC1 hexamers that are not cross-linked in the native GBM network and contain alpha3 NC1 monomers. (2) Alport allo-epitopes are accessible in the GBM network and map to the surface of the NC1 hexamer. (3) The GP epitopes of alpha3(IV)NC1 are nephritogenic and will induce pathogenic GP-like autoAbs in mice immunized with alpha3 NC1 monomers, chimeras containing the epitopes, and dissociated--but not native--GBM NC1 hexamers. (4) The specificity and pathogenicity of anti-GBM Abs are modulated by immune tolerance and by the association state (monomer vs. hexamer) of nephritogenic NC1 domains. It is anticipated that completion of these aims will provide a better understanding of the molecular etiology and pathogenesis of human GP disease and Alport post-transplant nephritis, will establish animal models that more closely resemble human anti-GBM disease, and may identify new molecular targets for prevention or therapeutic intervention.